Botulinum toxin type A heavy chain TD protein and application thereof

Abstract

The invention discloses a botulinum toxin type A heavy chain TD protein and application thereof. The amino acid sequence of the TD protein is as shown in SEQ ID No. 1, and the nucleotide sequence of the coding gene of the TD protein is as shown in SEQ ID No. 2. The botulinum toxin type A heavy chain TD protein is applied to preparation of anti-depression drugs, and is especially used for protecting neuronal functions and improving depression-like behaviors. The A-type botulinum toxin heavy chain TD protein effectively avoids the toxic effect of traditional A-type botulinum toxin, only effective fragments playing an anti-depression role in the A-type botulinum toxin heavy chain are extracted, and the effective anti-depression role is played by preparing small-molecule oligopeptide protein.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a type A botulinum toxin heavy chain TD protein and its application. The TD protein is used to prepare antidepressant drugs, especially for protecting neuronal function and improving depressive-like behavior. Background Technology

[0002] Depression is a common mental illness, primarily characterized by low mood, loss of interest, slowed thinking, lack of initiative, self-blame and guilt, poor appetite and sleep, and in severe cases, suicidal thoughts and behaviors. The incidence of depression is showing an upward trend year by year; according to data from the World Health Organization (WHO) in 2023, there were over 280 million people suffering from depression globally. The treatment outcomes for depression are not optimistic. The latest data published in *The Lancet Psychiatry* in 2021 showed that only 9.5% of respondents with depression received mental health services, and only 0.5% received adequate treatment. Furthermore, even standard antidepressant medications and psychotherapy are effective in only about 74% of cases, and approximately 30-60% of patients do not experience relief from depressive symptoms after medication treatment or even develop drug resistance.

[0003] Current antidepressants are classified according to their mechanism of action or chemical structure into selective serotonin reuptake inhibitors (SSRIs), serotonin and norepinephrine reuptake inhibitors (SNRIs), norepinephrine and specific serotonergic antidepressants (NaSSAs), tricyclic (TCAs) and tetracyclic antidepressants, monoamine oxidase inhibitors (MAOIs), etc. Most patients with depression experience symptom relief with treatment, but approximately 30% of patients do not respond or respond very little to adequate treatment with two or more antidepressants of different chemical structures at sufficient doses. With the emergence of drug resistance and side effects from classic antidepressants, there is an urgent need to find new antidepressants.

[0004] The gut microbiota is a vast community of microorganisms residing in the intestines of organisms, playing important physiological and pathological functions. With the development of sequencing technology, the role of the gut microbiota in diseases has been increasingly elucidated. Studies have shown that patients with depression exhibit gut microbiota dysbiosis, and fecal microbiota transplantation can influence the depressive phenotype, suggesting that the gut microbiota plays a role in the pathology of depression. The gut microbiota is dynamic and diverse, and capable of external regulation; therefore, rebalancing the gut microbiota has been considered a promising treatment for depression. Our study found a decreased abundance of Clostridium botulinum in the intestines of mice with depression, and this change was also observed in clinical samples of depression, suggesting that Clostridium botulinum may play an important role in improving depression.

[0005] Clostridium botulinum is a bacterium that releases dangerous toxins in anaerobic environments. Botulinum toxin type A is one of the most lethal substances known; its heavy chains target neurons, while its light chains cleave synaptic proteins, inhibiting nerve function and causing paralysis of respiratory and other muscles. However, trace amounts of botulinum toxin can be used to improve facial wrinkles, muscle spasms, and relieve pain. There are reports in the literature that facial injections of botulinum toxin can improve depressive mood in patients during clinical trials. Finzi first applied botulinum toxin type A to the treatment of depressive disorders in 2006, and research results showed that it could significantly alleviate symptoms in patients with depressive disorders. However, the efficacy of most patients gradually diminishes after a single injection, requiring multiple injections. Common adverse reactions mainly include localized weakness in adjacent muscles due to toxin diffusion to nearby muscles; a few patients may experience dizziness. The strong toxicity of botulinum toxin type A to the peripheral nervous system limits its antidepressant application.

[0006] Therefore, further research on botulinum toxin type A to obtain its core antidepressant domain and remove its application limitations is of practical significance and has good development prospects. Summary of the Invention

[0007] The purpose of this invention is to reveal the application of a type A botulinum toxin heavy chain TD protein in the preparation of antidepressant drugs.

[0008] This invention reveals that the abundance of *Clostridium botulinum* is downregulated in the gut microbiota of mice with depression and patients with depression. Supplementation with type A botulinum toxin improved depressive-like behaviors in mice with depression induced by various methods. Using the *Clostridium botulinum* heavy chain as a template, the TD, NRBD, and CRBD regions were amplified to construct an *E. coli* overexpression system, and the engineered *E. coli*::TD strain significantly improved the depressive phenotype in mice. The type A botulinum toxin heavy chain TD protein was prepared through protein purification, and this TD protein alleviated depressive-like behaviors in mice with depression. This invention uses engineered bacteria to decompose the structural domains of type A botulinum toxin, revealing that the TD domain of its heavy chain has a good antidepressant effect, and its application in the treatment of depression is unprecedented.

[0009] The specific solution of the present invention is as follows: A type A botulinum toxin heavy chain TD protein, the amino acid sequence of which is shown in SEQ ID No. 1.

[0010] The gene encoding the type A botulinum toxin heavy chain TD protein described herein has the nucleotide sequence shown in SEQ ID No. 2.

[0011] Engineered bacteria expressing the type A botulinum toxin heavy chain TD protein.

[0012] The application of the type A botulinum toxin heavy chain TD protein in the preparation of antidepressant drugs and drugs that protect neuronal function.

[0013] Furthermore, the drug is prepared by mixing the TD protein with excipients into any pharmaceutically acceptable dosage form.

[0014] Furthermore, the excipients include any one or more of sucrose, starch paste, solubilizer, and coating material.

[0015] Furthermore, the dosage forms include powder formulations, capsules, tablets, liquid pellets, or liquid injection formulations.

[0016] The gene has applications in the preparation of antidepressant drugs and drugs that protect neuronal function.

[0017] Application of the engineered bacteria in the preparation of antidepressant drugs and drugs that protect neuronal function.

[0018] The advantages and beneficial effects of this invention are: The type A botulinum toxin heavy chain TD protein described in this invention effectively avoids the toxic effects of traditional type A botulinum toxin. It extracts only the effective fragments of the type A botulinum toxin heavy chain that exert antidepressant effects, and achieves effective antidepressant effects by preparing small molecule short peptide proteins. Attached Figure Description

[0019] The present invention will be further described below with reference to the accompanying drawings and embodiments.

[0020] Figure 1 The abundance of Clostridium botulinum was reduced in the gut microbiota of patients with depression and mice with depression models. Figure 2 To improve the depressive phenotype induced by traditional depression modeling methods using BoNT / A; Figure 3 To improve the depressive phenotype in transgenic mice with BoNT / A; Figure 4 The effects of BoNT / A light chain or heavy chain Escherichia coli overexpression systems on depressive behavior in mice; Figure 5 The effect of the BoNT / A heavy chain TD Escherichia coli overexpression system on depressive behavior in mice; Figure 6 To improve the depressive phenotype in a mouse model of genetic depression by using BoNT / A heavy chain TD protein; Figure 7 To improve the depressive phenotype induced by traditional depression modeling methods in mice using BoNT / A heavy chain TD protein; Figure 8 BoNT / A heavy chain TD protein protects neuronal function.

[0021] BoNT / A indicates type A botulinum toxin. The same applies below. Detailed Implementation

[0022] The present invention will be further described below with reference to embodiments, but the scope of protection of the present invention is not limited thereto: The type A botulinum toxin heavy chain TD protein obtained in this invention is a 50 kDa protein fragment on the heavy chain of the non-toxic component of type A botulinum toxin, and its amino acid sequence is shown in SEQ ID No. 1. The gene encoding the type A botulinum toxin heavy chain TD protein is shown in SEQ ID No. 2. The TD protein restores depressive-like behaviors in various mouse models of depression.

[0023] Experimental Example 1: Analysis of the relative abundance of Clostridium botulinum in the gut microbiota of fecal samples from a clinical cohort of depression revealed a decrease in the abundance of Clostridium botulinum in the gut of patients with depression.

[0024] Through conditional knockout of the nervous system Chrna7 Gene( Chrna7 Nes A transgenic mouse model of depression was constructed, and feces were collected. DNA was extracted and sequenced using metagenomics. The relative abundance of gut microbiota in the feces of gene knockout mice and non-knockout mice was detected, and it was found that the abundance of Clostridium botulinum in the gut of the depression model mice was reduced compared with the control group.

[0025] Figure 1 The abundance of Clostridium botulinum in the gut microbiota of patients with depression and mice with depression models was reduced. Compared with the normal control group (HC), the abundance of Clostridium botulinum in the gut microbiota of patients with depression (MDD) was reduced. Figure 1 A). Chrna7 Nes Significant differences were found in the α- and β-diversity of the gut microbiota between mice and wild-type (WT) mice. Figure 1 BC). Compared to WT mice, Chrna7 Nes Differences were observed in the abundance of the gut microbiota at the genus level in mice, with Clostridium species being downregulated. Figure 1 D); At the bacterial species level, the abundance of Clostridium botulinum was significantly downregulated. The statistical plots were generated using the rank-sum test. The population cohort consisted of a control group of 155 individuals and a depression group of 156 individuals. Chrna7 Nes The number of model mice used for sequencing analysis was 8.

[0026] Experimental Example 2: Clostridium botulinum secretes BoNT / A, which plays a pathophysiological regulatory role. A mouse model of depression was established using traditional chronic unpredictable stress (CUMS). Male C57BL / 6J mice were randomly divided into a control group (n=12) and a CUMS group (n=18). The CUMS group was modeled using the stress protocol described in previous studies by Willner P and Zhao M et al. (Willner P, et al. Psychopharmacology (Berl).PMID: 3124165. Zhao M, et al.CellMetabolism.PMID: 38582087.), while the control group maintained normal feeding without external stress intervention.

[0027] Three weeks after modeling, nine CUMS mice were randomly selected and treated with either BoNT / A (0.3 U / time, every 3 days for 3 weeks) or an equal volume of saline via intraperitoneal injection. Behavioral experiments were conducted to assess the mice's depressive phenotype. The results showed that CUMS mice exhibited shorter dwell times in the central region of the open field test (OFT) and longer immobility times in the tail suspension test (TST) and forced swimming test (FST). BoNT / A significantly increased the dwell time in the central region of the open field test and reduced the immobility times in the tail suspension test and forced swimming test.

[0028] Thirty male C57BL / 6J mice were randomly divided into three groups: a control group (CON) (n=10), a corticosterone treatment group (CORT) (n=10), and a corticosterone + botulinum toxin type A treatment group (CORT+BoNT / A) (n=10). CON group mice were injected with olive oil containing 1% DMSO to eliminate carrier interference; CORT group mice received subcutaneous injections of 40 mg / kg corticosterone (dissolved in olive oil containing 1% DMSO) daily for 3 weeks; CORT+BoNT / A group mice received subcutaneous corticosterone injections and intraperitoneal injections of botulinum toxin type A (0.3 U / injection, every 3 days for 3 weeks). Behavioral experiments to assess the depressive phenotype in the mice revealed that CORT mice exhibited prolonged immobility in the tail suspension test and forced swimming test, while botulinum toxin type A significantly reduced immobility in these tests. These data indicate that botulinum toxin type A can improve depressive-like behaviors in traditional depression models in mice.

[0029] Figure 2BoNT / A improved the depressive phenotype induced by traditional methods of modeling depression in mice. CUMS-treated mice had shorter center dwell times in the open field (OFT) test and longer immobility times in the tail suspension test (TST) and forced swimming test (FST), and their phenotype was restored after BoNT / A replacement. Figure 2 AC. Mice treated with CORT showed shorter dwell times in the open field and prolonged immobility times in the tail suspension test (TST) and forced swimming test (FST). Immobility times in the open field remained unchanged after BoNT / A replacement, while immobility times in the tail suspension test and forced swimming test were restored. Figure 2 DF). Statistical graphs were generated using one-way ANOVA, with each group expressed as mean and standard deviation, and sample sizes ranging from 7 to 12.

[0030] Experimental Example 3: Conditional knockout in the nervous system Chrna7 Gene-derived transgenic mouse model of depression ( Chrna7 Nes ), and conditional knockout of the limbic cortex Chrna7 Gene-derived transgenic mouse model of depression ( Chrna7 ObRb The mice were treated with either BoNT / A (0.3 U / time, every 3 days for 3 weeks) or an equal volume of saline as controls. BoNT / A significantly increased the time mice spent in the central region of the open field test, reduced immobility time in the tail suspension and forced swimming tests, and increased grooming time in the sugar water splash test. These data indicate that botulinum toxin type A can improve depressive-like behaviors in a transgenic mouse model of depression.

[0031] Figure 3 BoNT / A improved the depressive phenotype in transgenic mice with depression. Compared with wild-type (WT) mice, Chrna7 Nes Mice exhibited shorter center dwell time in the open field (OFT) test, prolonged immobility time in the tail suspension test (TST) and forced swimming test (FST), and shorter grooming time in the sugar water splash test (SST). Their phenotype was restored after BoNT / A replacement. Figure 3 AD). Chrna7 ObRb Mice also exhibited shorter center dwell times in the open field (OFT) test, longer immobility times in the tail suspension test (TST) and forced swimming test (FST), and shorter grooming time in the sugar water splash test (SST). Their phenotype was restored after BoNT / A treatment. Figure 3 EH). Statistical graphs were generated using one-way ANOVA, with each group expressed as mean and standard deviation, and a sample size of 10-12.

[0032] Example 1: To identify the key structural domains responsible for the antidepressant effect of BoNT / A, it was divided into light and heavy chains, and the DNA was amplified separately, ligated into the PET-28a plasmid, and transformed into E. coli. The engineered bacteria were inoculated into liquid LB medium and amplified to an OD value of 0.8. Isopropyl-β-D-thiogalactoside (IPTG) was then used to induce the expression of either the light or heavy chain protein.

[0033] The light chain DNA sequence is SEQ ID No. 3 (1344bp): The heavy chain DNA (codon-optimized) sequence is SEQ ID No. 4 (2544bp): Table 1 LB medium formulation One week after administering combined antibiotics in drinking water, the bacterial culture overexpressing light or heavy chain proteins ( E.Coli ::LC or E.Coli ::HC) Gavage refeeding Chrna7 Nes Transgenic depression model mice (bacterial concentration 1×10⁻⁶) 8 CFU / ml, 0.2ml / dose, replenished every 3 days for 3 weeks.

[0034] Table 2 Combined antibiotic formulations Using behavioral testing to detect depressive phenotypes in mice, the results showed that supplementation E.Coli ::LC did not improve depressive-like behavior in mice, while complementation E.Coli ::HC can significantly improve depressive-like behavior in mice, indicating that the heavy chain of BoNT / A plays an antidepressant role.

[0035] Figure 4 Effects of BoNT / A light or heavy chain Escherichia coli overexpression system on depressive behavior in mice. Chrna7 Nes Mice receiving E.Coli ::LC replenishment did not improve the dwell time in the central region of the open field test (OFT), the immobility time in the suspended tail test (TST) and forced swimming test (FST), or the grooming time in the sugar water splash test (SST). Figure 4 AD); and Chrna7 Nes Mice receiving E.Coli ::HC replenishment restored the dwell time in the central region of the open field test, the immobility time in the suspended tail test (TST) and forced swimming test (FST), and the grooming time in the sugar water splash test (SST). Figure 4 EH). Statistical graphs were generated using one-way ANOVA, with each group expressed as mean and standard deviation, and a sample size of 10-12.

[0036] Example 2: The heavy chain was further divided into three domains: TD, NRBD, and CRBD. These domains were overexpressed in E. coli and then administered to transgenic depression model mice via gavage after combined antibiotic treatment. Chrna7 Nes (Bacterial concentration is 1×10) 8 CFU / ml, 0.2ml / dose, replenished every 3 days for 3 weeks.

[0037] The TD fragment DNA (codon-optimized) sequence is SEQ ID No. 2 (1266bp): The NRBD fragment DNA (codon-optimized) sequence is SEQ ID No. 5 (666bp): The CRBD fragment DNA (codon-optimized) sequence is SEQ ID No. 6 (612bp): After reinjection, behavioral tests were performed on the mice to detect depressive phenotypes. The results showed that reinjection... E.Coli ::TD can improve depressive-like behavior in mice. This suggests that BoNT / A exerts its antidepressant effect through the heavy chain TD fragment.

[0038] Figure 5 : TD fragment of BoNT / A heavy chain ( E.Coli ::TD) improves the depressive phenotype in a mouse model of depression.

[0039] Chrna7 Nes Mice receiving E.Coli After reintroduction of ::TD-overexpressing engineered bacteria, the residence time in the central region of the open field (OFT) assay, the immobility time in the suspended tail (TST) and forced swimming (FST) assays, and the grooming time in the sugar water splash (SST) assay were improved; while E.Coli ::NRBD、 E.Coli ::CRBD overexpression of engineered bacteria had no effect ( Figure 5 (AD). The statistical graphs were generated using one-way ANOVA, with each group represented by the mean and standard deviation. The sample size was 10-12.

[0040] Example 3: Peptide drugs have been applied in the research of various diseases. In order to further clarify the role of BoNT / A heavy chain TD protein, we will... E.Coli After TD amplification and induced expression, the TD protein was purified using a nickel ion column, and the purified TD protein was intraperitoneally injected back into the body. Chrna7Nes Transgenic depressed mouse model (injection concentration 100ug / 0.2ml, PBS solvent, 100ug / time, treatment every 3 days for 3 weeks). Results showed that purified BoNT / A heavy chain TD protein significantly improved depressive-like phenotypes in various classic behavioral experiments of depression, including open field, tail suspension, forced swimming, and sucrose splashing, demonstrating a good antidepressant effect.

[0041] Figure 6 BoNT / A heavy chain TD protein improves the depressive phenotype in a mouse model of depression.

[0042] Chrna7 Nes After receiving TD protein rehydration, the mice recovered their time spent in the open field central region (OFT), immobility time in the tail suspension test (TST) and forced swimming test (FST), and grooming time in the sugar water splash test (SST). Figure 6 (AD). The statistical graphs were generated using one-way ANOVA, with each group represented by the mean and standard deviation. The sample size was 10-12.

[0043] Example 4: We also investigated the effects of BoNT / A heavy chain TD protein on the depressive phenotype in classic stress-induced and drug-induced (i.e., CUMS and CORT) mouse models of depression. We found that TD protein significantly improved depressive-like behaviors in CUMS model mice in open field, tail suspension and forced swimming tests, and was also effective in the recovery and treatment of CORT-induced depression.

[0044] Figure 7 BoNT / A heavy chain TD protein improves the depressive phenotype induced in mice by traditional methods of modeling depression.

[0045] In CUMS and CORT model mice, the dwell time in the central region of the open field test (OFT), the immobility time in the tail suspension test (TST), and the forced swimming test (FST) were all restored after TD protein recompensation. Figure 7 (AF). Statistical graphs were generated using one-way ANOVA, with each group expressed as mean and standard deviation, and a sample size of 10-12.

[0046] Example 5: PSD95 is involved in synaptic plasticity, neurotransmitter receptor anchoring (such as NMDA and AMPA receptors), and signal transduction. Restoring PSD95 expression or function may be one of the potential directions for developing novel antidepressant strategies. The detection of PSD95 after treatment with BoNT / A heavy chain TD protein... Chrna7 NesThe expression of PSD95 in the brain tissue of transgenic depressed mouse models was investigated, and it was found that TD protein significantly upregulated the expression of PSD95 protein in the brains of depressed mouse models. This suggests that TD protein has a protective effect on neuronal function and can improve the phenotype of depression.

[0047] Figure 8 BoNT / A heavy chain TD protein protects neuronal function.

[0048] Chrna7 Nes Mouse PSD95 protein expression was downregulated, and TD protein treatment restored PSD95 protein expression. Figure 8 ).

[0049] Finally, it should be noted that the above description is only used to illustrate the technical solutions of the present invention and is not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the present invention.

Claims

1. A type A botulinum toxin heavy chain TD protein, characterized in that: Its amino acid sequence is shown in SEQ ID No.

1.

2. The gene encoding the type A botulinum toxin heavy chain TD protein as described in claim 1.

3. The gene according to claim 2, characterized in that: The nucleotide sequence of the gene is shown in SEQ ID No.

2.

4. An engineered bacterium expressing the type A botulinum toxin heavy chain TD protein as described in claim 1.

5. The use of the type A botulinum toxin heavy chain TD protein as described in claim 1 in the preparation of antidepressant drugs and drugs that protect neuronal function.

6. The application according to claim 5, characterized in that: The drug is prepared by mixing the TD protein with excipients into any pharmaceutically acceptable dosage form.

7. The application according to claim 6, characterized in that: The excipients include any one or more of sucrose, starch paste, solubilizer, and coating material.

8. The application according to claim 6, characterized in that: The dosage forms include powder formulations, capsules, tablets, liquid pellets, or liquid injection formulations.

9. The use of the gene described in claim 2 in the preparation of antidepressant drugs and drugs that protect neuronal function.

10. The use of the engineered bacteria according to claim 4 in the preparation of antidepressant drugs and drugs that protect neuronal function.

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